Empiric vs Preemptive Antifungal Strategy in High-Risk Neutropenic Patients on Fluconazole Prophylaxis: A Randomized Trial of the European Organization for Research and Treatment of Cancer

Abstract Background Empiric antifungal therapy is considered the standard of care for high-risk neutropenic patients with persistent fever. The impact of a preemptive, diagnostic-driven approach based on galactomannan screening and chest computed tomography scan on demand on survival and on the risk of invasive fungal disease (IFD) during the first weeks of high-risk neutropenia is unknown. Methods Patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) and allogeneic hematopoietic cell transplant recipients were randomly assigned to receive caspofungin empirically (arm A) or preemptively (arm B), while receiving fluconazole 400 mg daily prophylactically. The primary end point of this noninferiority study was overall survival (OS) 42 days after randomization. Results Of 556 patients recruited, 549 were eligible: 275 in arm A and 274 in arm B. Eighty percent of the patients had AML or MDS requiring high-dose chemotherapy, and 93% of them were in the first induction phase. At day 42, the OS was not inferior in arm B (96.7%; 95% confidence interval [CI], 93.8%–98.3%) when compared with arm A (93.1%; 95% CI, 89.3%–95.5%). The rates of IFDs at day 84 were not significantly different, 7.7% (95% CI, 4.5%–10.8%) in arm B vs 6.6% (95% CI, 3.6%–9.5%) in arm A. The rate of patients who received caspofungin was significantly lower in arm B (27%) than in arm A (63%; P < .001). Conclusions The preemptive antifungal strategy was safe for high-risk neutropenic patients given fluconazole as prophylaxis, halving the number of patients receiving antifungals without excess mortality or IFDs. Clinical Trials Registration. NCT01288378; EudraCT 2010-020814-27.

guidelines [3,4]. However, empiric use of the recommended antifungals liposomal amphotericin B and caspofungin [5,6] most likely leads to overtreatment with increased toxicity and costs.
Previously, both strategies were compared in the randomized PREVERT study [23]. However, there were too few patients with prolonged neutropenia to rule out the noninferiority of survival with the preemptive strategy. Therefore, the Infectious Diseases Group and the Acute Leukemia Group of the European Organization for Research and Treatment of Cancer (EORTC) initiated this new trial with overall survival as the primary end point.

Study Design and Participants
The EORTC 65091-06093 study was an open-label, phase 3, randomized, parallel, multicenter, strategy trial comparing the efficacy and safety of a fever-driven antifungal approach (empiric, arm A) to a diagnostic-driven approach (preemptive, arm B) in neutropenic patients at high risk of developing IFD.
We recruited patients aged ≥18 years who were scheduled for remission-induction chemotherapy for newly diagnosed AML or MDS or in first relapse after remission of at least 6 months, or to start a myeloablative conditioning regimen [26] for a first allogeneic HCT. The main exclusion criteria were clinically documented pneumonia, uncontrolled infection, or previous IFD. Detailed eligibility criteria are provided in the Supplementary File S1.

Trial Oversight
The trial was sponsored by the EORTC and funded by Merck Sharp & Dohme Corp, a subsidiary of Merck & Co, Inc, Kenilworth, NJ, which also provided caspofungin but had no further role in the trial or in writing the manuscript. The trial statisticians performed the analyses and vouched for the integrity and validity of the analyses. The authors affirm that the trial was conducted as specified in the protocol and agreed with the final manuscript and approved it for publication.
The trial was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice guidelines and was approved by the institutional review board and ethic committee at each center. All patients provided written informed consent before undergoing any trial-specific procedures. A data review committee (DRC) reviewed eligibility criteria, compliance with the protocol, criteria for IFD, and causes of death. A blinded radiologist reviewed all the CT scans with no or inconclusive reports.

End Points
The primary end point was overall survival (OS) 42 days after randomization. Key prespecified secondary end points assessed at day 42 and day 84 after randomization included OS, rates of proven or probable IFD (using the 2008 EORTC/National Institute of Allergy and Infectious Diseases Mycoses Study Group (MSG) definitions [27]), compliance with the allocated treatment arm, survival free of proven or probable IFD, number of days of caspofungin administration, and safety. Adverse events (AEs) and serious AEs (SAEs) were assessed according to Common Terminology Criteria for Adverse Events criteria v4.0.

Randomization and Data Collection
Eligible patients underwent central randomization in a 1:1 ratio using the EORTC online system, stratified according to trial site, allogeneic HCT, and the use of laminar-airflow or highefficiency particulate-air filtered rooms. Randomization was done within 3 days after the start of chemotherapy or conditioning regimen.
Definitions of major protocol violations are listed in the Supplementary File S2.

Arm A (Empiric Arm)
Patients allocated to arm A were given caspofungin either for unexplained fever after 4 days of broad-spectrum antibacterials or for a new febrile episode more than 2 days after resolution of the first episode while continuing broad-spectrum antibacterials ( Figure 1). Patients allocated to this study arm were not screened for blood galactomannan before starting caspofungin.

Arm B (Preemptive Arm)
Patients in arm B were screened twice weekly on site for blood galactomannan with the enzyme immunoassay (Platelia, Bio-Rad Laboratories, Marnes-La-Coquette, France), according to the manufacturer's instructions. A positive galactomannan assay (optical density index above 0.5), a new pulmonary infiltrate on chest X ray, or the recovery of Aspergillus in sputum prompted a chest CT scan. Patients were given caspofungin when there was a single positive galactomannan test result (even when asymptomatic), a new pulmonary infiltrate on chest X ray and IFD could not be readily excluded per the investigator's judgment, a new pulmonary infiltrate on chest CT scan consistent with an IFD (a nodule, with or without a halo; a cavity; an air crescent sign), or/and the recovery of Aspergillus species by culture from sputum [27].

Study Procedures
All patients received fluconazole 400 mg/day to prevent Candida infection and remained in the hospital for the duration of neutropenia. All patients who developed a first episode of febrile neutropenia (temperature ≥ 38.3°C once or ≥ 38.0°C twice consecutively with an absolute neutrophil count [ANC] <500 neutrophils/mm 3 or expected to fall within 48 hours) underwent a diagnostic workup with clinical examination, chest X ray, or chest CT scan according to institutional practice; at least 2 separate sets of blood cultures; and appropriate specimens from any other potential sites of infection (as clinically indicated) for microbiology before starting broad-spectrum antibacterials according to local standards and consistent with the Infectious Diseases Society of America guidelines [3].
Once the study arm-specific criteria for starting caspofungin treatment were met, additional blood cultures were taken in both arms, as well as appropriate cultures from other sites, whenever clinically indicated. A chest X ray was ordered for patients in arm A, and a chest CT scan was ordered for patients in arm B. Following the initiation of caspofungin, blood samples for the detection of galactomannan were collected twice weekly in both study arms. Additional examinations, including cultures, imaging, bronchoscopy with bronchoalveolar lavage, and/or needle aspirates or biopsies, were performed on clinical indication.

Caspofungin
Caspofungin was given at a loading dose of 70 mg on day 1, thereafter 50 mg/day (70 mg if body weight exceeded 80 kg) until neutrophil recovery (ANC ≥ 500 neutrophils/mm 3 ) or until the diagnosis of a proven or probable IFD, whichever occurred first. Fluconazole prophylaxis was stopped, and no other systemic antifungal was allowed during administration of caspofungin. In case of proven or probable IFD, caspofungin was stopped and further antifungal therapy was given according to local guidelines.

Statistical Analyses
Patients were considered eligible if they satisfied all the entry criteria and met none of the exclusion criteria. All eligible patients constituted the modified intention-to-treat (mITT) population and were included in the primary analysis. The per-protocol (PP) population included only cases without any major protocol violation as defined by the DRC.
Our aim in this study was to show that the overall survival 42 days after randomization of the preemptive strategy (arm B) was not inferior to that of the empiric strategy (arm A). Every death was taken into consideration, regardless of the cause, for assessing the primary end point.
We estimated an 87% survival rate at day 42 in arm A based on the results of high-risk patients receiving fluconazole prophylaxis [28]. The greatest relative risk for death acceptable for the noninferiority of arm B was set at 1.62. With a power of 80% for rejecting the null hypothesis, we calculated that 556 patients were needed for the study.
The survival rates at day 42 were estimated in each treatment arm using Kaplan-Meier estimates, and we calculated the survival rate at day 42 of each arm and considered arm B as noninferior to arm A if the ratio of the upper bound 95% confidence interval (CI) for arm B/the lower bound 95% CI of arm A was less than 1.62.
The safety analysis included all randomized patients with the worst degree of toxicity measured between randomization and day 84 being reported. Only the rates of patients developing at least 1 grade 3/4 AE or at least 1 SAE in each randomization arm are reported with 95% CIs.
A planned interim analysis was done by the EORTC Independent Data Monitoring Committee (IDMC) in February 2014 when 263 patients had a follow-up ≥ 42 days. The aim was to determine whether the survival rate in the empiric arm A was in the expected range. The survival rate in the control arm was higher than anticipated (94%; 95% CI, 90%-98%). However, the IDMC recommended that the sample size not be increased as the noninferiority margin that was chosen was stricter than that chosen for the PREVERT trial [23]. In addition, increasing the sample size would have jeopardized recruitment, delayed the trial results, and increased costs.  Table 1.

Invasive Fungal Diseases
The rates of proven and probable IFD within 84 days after randomization were 7.7% in arm B and 6.6% in arm A (P = .61; Table 2) in the mITT population. There was no statistically significant difference in the IFD rates between the subgroups of AML/MDS patients and the HCT recipient group. Among the 39 proven or probable IFDs observed, 33 (84.6%) occurred before day 42. Eight of the 11 proven cases and 25 of the 28 probable cases were diagnosed within 42 days after randomization; the remainder were diagnosed between day 42 and day 84. Most proven IFDs were due to Candida, and all probable IFDs were aspergillosis.

Antifungal Therapies
The rate at which mITT patients received caspofungin treatment according to the randomized strategy was 27% in arm B vs 63% in arm A (P < .001), but with no difference in the caspofungin treatment duration ( Table 3). The rates of administration of other antifungals were similar between arms. The number of patients who started preemptive caspofungin on the basis of each triggering criterion (or combination of criteria) is summarized in Supplementary Table 5.

Safety
The rates for patients experiencing at least 1 grade 3, 4, or 5 AE or at least 1 SAE were not different between arms (Supplementary File S6).

DISCUSSION
This study shows that a preemptive antifungal strategy that includes twice weekly galactomannan screening and CT scan on demand does not prejudice the overall survival of adults with prolonged neutropenia who are at high risk for IFD while receiving fluconazole prophylaxis. In addition, is the strategy is not associated with an increased risk of proven or probable IFD. Indeed, the strategy reduces the use of antifungals by half, which should prove cost-saving. This study adds important information to previous trials, especially the French PREVERT study that was not sufficiently powered to establish the noninferiority of the preemptive strategy in the subgroup of patients receiving AML induction chemotherapy [23].
Several differences should be noted between the 2 trials. The present study was exclusively focused on long-term neutropenia; allogeneic HCT recipients were included; all patients received fluconazole prophylaxis; the IFDs were defined according to the EORTC/MSG 2008 consensus definitions [27], not the earlier version [29]; and caspofungin was used exclusively for both empiric and preemptive therapy. In the PREVERT study, the rate of IFDs in the AML-induction group (with a median duration of neutropenia of 26 days) that was preemptively managed was 16.4% and significantly higher than the rate of the empiric group (3.8%). Although this difference could partly be explained by more diagnostic procedures used in the preemptive arm, it could also be due to use of the original 2002 EORTC/MSG definitions rather than the revised 2008 definitions. It could also be attributed to an increased risk for IFD due to the administration of antifungals to fewer patients for a shorter time in the preemptive strategy.
Others also reported an excess risk for IFD with a preemptive strategy [16,21]. Even though an AML patient may initially survive the IFD, having an IFD impacts the long-term outcome  Documented by positive culture of BAL 3 1
due to the risk of IFD recurrence, leading to modifying or postponing subsequent courses [30]. Hence, relying only on survival of less than 3 months could miss the consequences of IFD on the final outcome. In the present study, we did not observe any excess IFDs with the experimental strategy, so this fear can be allayed. With an IFD rate of 7% in the AML group, our results are consistent with the 8% rate in the Prospective Invase Mould Disease Audit (PIMDA) study [31]. Similarly, our IFD rate of 4.5% assessed within 12 weeks after starting the conditioning regimen in HCT recipients is consistent with recent data showing that currently two-thirds of the aspergillosis cases observed after allogeneic HCT occur after day 100 post-transplant [32].
The preemptive strategy is applicable as long as the center uses routine screening of galactomannan, has ready access to CT scans, and the costs are balanced by the reduced use of antifungals. Today, many centers use some hybrid strategy, mixing empiric administration of antifungals and a biomarker or imaging screening, which adds the cost of overuse of antifungals to the costs of biologic screening and CT scan.
Our study has several strengths. First, we chose survival as a hard and objective primary end point, considering that survival is a prerequisite for a favorable outcome. This end point highlights the need for the best strategies to be used during high-risk neutropenia. Second, our groups were well balanced in terms of AML risk and neutropenia duration, precluding any impact of these parameters on survival. Third, we assessed only proven and probable IFDs and did not consider possible IFDs where fungal causality is less certain. Last, the size of the AML cohort allowed us to be confident of the overall results and to generalize them to such patients as a whole.
No study is free of limitations. First, the comparison of 2 strategies with different diagnostic workup and triggering factors for antifungal administration made a blinded design impossible to apply. Second, we chose not to use antimold prophylaxis because it lowers the sensitivity of galactomannan for screening. So, while our preemptive strategy would not be suitable for those centers that do use antimold prophylaxis [33], either strategy would be useful to those centers that do not use it or in patients who cannot continue on mold-active azole prophylaxis due to AEs or clinically important drugdrug interactions.
Empiric antifungal therapy has been the gold standard for managing IFD in neutropenic patients and will remain so for centers with limited diagnostic resources that rely on a clinically driven approach. The results of our study now provide a viable alternative.

Supplementary Data
Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.